Enzyme-mediated modification of fibrin for tissue engineering: fibrin formulations with peptides

Abstract

Heparin-binding regions of several proteins, such as neural cell adhesion molecule, fibronectin, laminin, midkine, and anti-thrombin III have been shown to promote neurite extension on two-dimensional surfaces. The effect of heparin-binding peptides on neurite extension through three-dimensional matrices was investigated by culturing embryonic chick dorsal root ganglia (DRG) within fibrin gels containing chemically attached heparin-binding peptide (HBP). The length of neurites within fibrin gels containing cross-linked HBP was increased by more than 70% over extension through fibrin gels containing no peptide. The HBP sequence of antithrombin III was incorporated into the fibrin gel as the C-terminal domain of a bidomian, chimeric peptide; the N-terminal second domain of this peptide contained the ∀2-plasmin inhibitor substrate for Factor XIIIa. Factor XIIIa, a transglutaminase, was used to chemically attach the HBP-containing chimeric peptide to the fibrin gels during polymerization. The amount of HBP cross-linked into the fibrin gels was determined, after degradation by plasmin using gel permeation chromatography, to be approximately 8 moles of peptide per mole fibrinogen. A peptide (HBP), where the cross-linking glutamine was replaced with glycine, showed no increase in extension in comparison with fibrin gels. The additional of heparin to the gel percursors resulted in no increase in neurite extension in comparison with fibrin gels. HBPs promote neurite extension by binding to cell surface proteoglycans on the DRG.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of application Ser. No. 09 / 141,770, filed Aug. 27, 1998 now abandoned, entitled “Enzyme-Mediated Modification of Fibrin for Tissue Engineering: Fibrin Formulations with Peptides”, by Jeffrey A. Hubbell, Jason C. Schense and Shelly E. Sakiyama.FILED OF THE INVENTION[0002]The present invention relates generally to the fields of materials useful in the support of tissue and cell incorporation and growth. More particularly, it concerns particular substrates of protein, such as fibrin, to which bioactive peptides and combinations of peptides are attached.BACKGROUND OF THE INVENTION[0003]The active domain of many proteins may in some cases be mimicked at least in part through the use of short peptide sequences derived from the active site of the protein (Massia and Hubbell, 1991, Yamada, 1991). Through this method, the activity of a specific protein can be conferred to an otherwise nonactive surface or matrix....

AI Technical Summary

Benefits of technology

[0011]All of the above protein and gel matrices may be further defined as three-dimensional matrices. Surprisingly, these three-dimensional matrices modified to incl

Problems solved by technology

However, if the RGD (SEQ ID NO:2) density is too low, then there is not enough traction for these cells to effectively migrate across the surface, leading to a maximal migration rate at a moderate surface concentration of peptide.

(DiMilla, et al., 1991) Unfortunately, there is not very much research of the concentration dependent effect of these peptides in

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